Microorganisms that associate with higher eucaryotes, be they commensals, symbionts or pathogens, are generally well-adapted to the niche provided by their host. Included in their genetic repertoire are systems that allow them to sense host factors and to use these signals to invoke behavioral responses appropriate to the interaction. It also has become clear recently that the individual cells of a microbial community can communicate with each other via chemical signals. This communication system can lead to genetic changes that are favorable to the microbe on a community scale. One such microbe-microbe signalling mechanism, called autoinduction, recently was found to play a key role in regulating the interactions between certain pathogenic bacteria and their animal and plant hosts. In this system, the bacteria secrete a small homoserine lactone derivative called autoinducer (HSL-AI). Induction of genes required for virulence is dependent upon the accumulation of this signal molecule. If this is blocked, the bacteria generally cannot cause disease. Thus, if we understood how this signalling process works, we conceivably could develop treatments that specifically block the pathway. As antibiotics become less effective, we must consider all options in our efforts to develop new antimicrobial agents. The plant pathogen, Agrobacterium tumefaciens is an excellent organism in which to study microbe-host and microbe-microbe signalling systems. As part of the interaction with its host, A. tumefaciens senses a set of specific signals produced by the diseased plant and responds by elaborating its own HSL-AI mediated cell-cell signalling system. When this second messenger accumulates to a critical level, it triggers a change in the bacterial community leading to the conjugal transfer of a virulence plasmid from the bacterial pathogen to non-pathogenic relatives. Thus, the signal potentiates the spread of the virulence plasmid among the agrobacteria in the soil. The HSL-AIs function as co-inducers to gene activator proteins. Many different microorganisms utilize these HSL messengers, but a given bacterium may recognize only its cognate signal. Specificity is conferred by relatively minor differences in the structures of the AIs. The basis for this specificity at the molecular level is not known. Nor is the mechanism by which the HSL-AI acts as a transcriptional co-inducer. We propose to examine some of these problems in the Agrobacterium system. The basis for HSL-M specificity will be tested by domain switching experiments, and by isolation of mutants that can respond to other, non- cognate HSLs. The influence of the HSL on the ability of the activator protein to bind DNA will be assessed using the in vivo P22 challenge phage system. The mechanism by which the amino acid methionine specifically blocks the signal pathway will be determined by genetic analysis of resistant mutants, as well as physiological analysis of HSL-AI diffusion into and out of the cells. The role of a newly discovered component, ModA, in attenuating signal sensing will be examined by testing the ability of this protein to interact with the activator protein. Finally, the molecular mechanism by which the plant signal activates the HSL-AI second message pathway resulting in gene induction will be examined by Northern analysis, promoter mapping, and transposon mutagenesis.